The present invention relates to a hematology blood control and, more particularly, to a hematology control made from human leukocytes and a method for the preparation of the control.
Instruments for the analysis of blood components and chemistry have been used for many years. The accuracy and sensitivity of these hematology instruments have steadily advanced. The early forms of hematology instrumentation have evolved to relatively complex machines that analyze the discreet components of blood based upon the intricate and subtle characteristics of its components.
The most recent iteration in automated hematology instrumentation has been the multi-part analysis of human white cells, in addition to the detection of red blood cells and platelets. The white cell populations typically include lymphocytes, monocytes, neutrophils, basophils, and eosinophils. The methods for blood cell analysis involve the electrical and optical properties of each type of blood cell. The Beckman-Coulter(trademark) five-part white cell analysis instrument uses three different types of technologies, which include electrical impedance, a DC mathematical manipulation called conductance, using a low voltage DC (direct current) measurement, Rf (radio frequency modulation), and laser technology which includes light scatter and light absorption. The Rf measurement is typically used with the DC low frequency measurement to create a parameter called opacity which is a calculation of Rf divided by DC. Instruments by other manufacturers, such as Abbott Diagnostics(trademark), Technicon(trademark), and TOA(trademark), use a combination of electrical impedance, DC conductance and/or laser technology, Rf, depolarized 90xc2x0 angle light scatter, and/or light absorption. Although the basic types of electronic technology may appear the same, each manufacturer has a unique implementation for the instrument hardware and software that is required to analyze blood cells. The individual implementations of this technology by the various manufacturers have resulted in a wide array of reagents and methodology for each specific instrument of each manufacturer, thereby increasing the complexity and expense of their use. There is no one reagent or methodology that can be used with a plurality of instruments.
In order to ensure that a hematology instrument is working properly, it has been mandated by governments that there be a method to verify the integrity of the instrument using a blood control. The control should contain particles that represent all of the cellular elements of fresh blood, as well as a liquid component that serves as a suspending media similar to the function of human plasma. This synthetic plasma usually contains components that are the same as or function the same as native plasma. The components of synthetic plasma include inorganic salts, organic and/or inorganic buffers, and a viscous material for maintaining homeostasis similar to the plasma proteins. The manufacturer of a control provides all of the critical values such as cell count, cell size, and cell type. The control material should have sufficient shelf life to be used for days, weeks, or months to ensure the consistency of instrument performance over time.
The method for preparing a hematology control is dependent on the hardware and software design of the specific instrument in which the control is to be used, as well as the requirements for extended shelf life. Particles in blood control products that work like human white cells, red blood cells, or platelets on a Coulter(trademark) type instrument may not work effectively on other instruments, such as instruments manufactured by Abbott Laboratories(trademark), Technicon(trademark), or TOA(trademark) instruments.
Moreover, because these particles are usually modified from various types of blood cells, they do not behave like living native fresh blood cells. Consequently, human white cells fixed with a cross-linking agent like glutaraldehyde may behave like a neutrophil on an Abbott(trademark) instrument, but behave like cellular debris on a Coulter(trademark) instrument. Specially treated and cross-linked red blood cells from non-mammalian vertebrates may look like mononuclear cells on one type of hematology instrument and look like lymphocytes on another.
The control of the present invention is achieved using a gentle chemical removal of red cells, leaving an intact white cell preparation. The preservation steps, namely the use of cross-linking agents such as aldehydes, involves a step-wise process starting with very low concentrations of a cross-linking agent. The fixation part of the process outlined in this disclosure has also been applied to non-mammalian blood cells and therefor would appear to be a universal procedure for preparing all types of vertebrate blood cells. The examples below include small as well as large volume processes. It has been found that the concentration and timing elements must be adjusted based on the volume of the reaction mixture. Thus, a final volume of 15 mL will require different proportions of elements and different timing steps compared to a 1 liter batch. For example, it is possible to perform a 15 second centrifugation with a 15 mL tube process but this 15 second step is essentially impossible with a 1 L bottle batch. The selective use of lytic agents and the unique fixation process eliminates the need for supportive reagents as cholesterol, which have been found to be necessary for the successful display of preserved human white cells.
The basic components for the preparation of the control of the present invention include the following:
A quantity of source leukocytes between 0-5 days old, preferably one day old, stored in a styrofoam or other insulated container with sufficient cold packs to insure the units are cool but not cold. Overnight storage at 4xc2x0 C. will sufficiently alter the quality of the white cells or potentially require modifications in the processing of the blood.
A lytic agent including of any one or a combination of the following:
an organic acid such as formic, acetic, and propionic acids. The preferred acid is propionic acid.
a quantity of saponin in water. Suitable types of saponin include, but are not limited to, Sapindus and Quillaja. The preferred saponin is Sapindus.
A quench made with inorganic salts including one or a combination of the following:
Carbonate buffered, similar to that used in certain commercial products, such as Hematronix, Inc. Diff Pak(trademark) containing sodium carbonate, sodium sulfate, and sodium chloride.
Buffer free sodium sulfate and sodium chloride salts.
Buffer free sodium sulfate only
Buffer free sodium chloride only
Sodium phosphate only
Any of the above salt solutions with cross-linking agent.
A post-lytic hypotonic fixing reagent including one or more of the following:
Low osmolarity salt, such as diluted M-Ringers, an xcex1-naphtol based salt solution, or diluted mammalian balanced salt solution as Osmocel(copyright).
a low carbon number glycol, such as propylene glycol
a cross-linking agent, such as an aldehyde like glutaraldehyde.
An additional post-lytic reagent including one or more of the following:
a cross-linking agent, such as an aldehyde like glutaraldehyde.
The preferred method for achieving the control of the present invention includes the gentle removal of non-white cell components from commercially available Source Leukocytes and the gentle step-wise preservation of the remaining white cells. The gentle removal of non-white cell components (i.e. red blood cells) from Source Leukocytes requires a lytic agent which preferentially removes blood cells. The preferred lytic agent includes a specific type of saponin called Sapindus. Other agents, such as short chain fatty acids with or without saponin, are also effective. Optimization of the process with saponin rather than a short chain fatty acid showed better performance when comparing weak versus strong Coulter instrument reagents.
The time of Source Leukocyte exposure to lyse and the volume of lyse are directly related to the size (volume) of the prepared white cells. Increased lyse time destroys non-neutrophil particles (i.e. lymphocytes, monocytes, and eosinophils) faster than neutrophil particles. The red blood cell count in the Source Leukocyte material has some effect (slight increase in white cell volume with increased red cell count) on the quality of the preserved white cells when using low red cell count versus high red cell count in the Source Leukocyte units.
After lytic treatment of the Source Leukocytes, the remaining white cells are exposed to a hyperosmotic salt solution (i.e. quench) containing no fixative or a low to moderate concentration of fixative, in the range of 0.1% to 0.5%. The salt solution raises the ionic strength to normal or near normal concentration and it prevents protein agglutination and controls the cell size and laser light scatter. The low concentration of cross-linking agent in the quench or added soon after the quench, provides a mild stabilization of the protein moieties of the white cells.
The cells in lyse, quench, and glutaraldehyde are incubated for a short time, approximately one hour or less. Then the lytic/quench/fix solution is replaced with a hyposmotic solution containing a second round of low to moderate cross-linking agent, from about 0.5 to 10% glutaraldehyde, for 1-8 days at room temperature. It is preferable that the solution be kept at approximately 37xc2x0 C. for a period of time, preferably 2 and one-half hours, to maintain long term stability of cellular characteristics as observed on a Coulter(trademark) 5-part WBC analyzer. Incremental decreases in the salt concentration will increase the particle volume and decrease Coulter(trademark) Scatter. Incremental increases in the salt concentration will decrease particle volume and increase Coulter(trademark) Scatter. Adding a cross-linking agent after exposure to the hyposmotic salt solutions results in a slightly higher volume than adding directly to the hyposmotic salt solution. If a larger volume is required, adding the cross-linking agent to the hyposmotic solution will provide an incremental increase in volume.
A third fixation may be applied using moderate to high glutaraldehyde fixation, from about 1-20%. The fixative is typically added directly to the supernatant rather than washing the cells as part of the third (tertiary) fixation.
The general procedure for treating human white cells includes a very low concentration of fixative after red cell lysis. Successive fixations involve an increase in the concentration of cross-linking agent by geometric or preferred log concentrations. The successive fixation steps are performed to maintain the characteristics of non-treated white cells and to extend the stability of the treated white cells. For example, if the first fix is at 0.1% glutaradehyde, the second fixation would be 1% and the third would be 10%. In concert with the progressive fixation steps, it has been found that elevated heating of the secondary fixed cells for the first 0.25-2 hours of exposure results in larger more stable particles. To provide added stability, a third fixation step is provided at a higher concentration of cross-linking agent than the first and second fixation steps.
Finally, the fixed white cells are washed in a hyposmotic solution then resuspended and stored in an hyposmotic or isosmotic solution for 1-30 days then added to preserved red blood cells in a synthetic plasma.
Decreasing the ionic strength from 60% Ringers to 20% Ringers will increase the volume depending on the strength of the cross-linking agent.
Some of the fixation steps, such as the first and third, are not required to obtain acceptable results, but do provide better quality and better long term stability. The gentle removal of non-white cell elements, the timing of the lysis, the use of lyse concentration and lyse volume, and the use of low to high concentrations of cross-linking agents during the process provide the desired stability and quality to be maintained in a commercial blood control red blood cell suspension.
Another agent important to the process is a low molecular weight glycol, such as propylene glycol, which has been found to selectively preserve white cells that have unique properties depending on the assay instrument. For example, propylene glycol was found to effect the display of particles in the monocyte region on the Coulter(trademark) Model MAXM(trademark) where the same preparation on the Abbott(trademark) 3500(trademark) did not display the particles in the monocyte region. If the propylene glycol was removed from the preparation of Source Leukocytes, there was no display of particles in the monocyte region on the Coulter(trademark) instrument, but they were present with the Abbott(trademark) instrument.
The preferred method for preparing a hematology control in accordance with the present invention includes the following:
the time of cell exposure to lyse is reduced by optimizing:
the concentration of the lytic agent
having the largest practical ratio of lyse to Source Leukocytes.
the cells were given a preliminary fix as quickly as possible (elevated heat) with the lowest concentration of cross-linking agent (e.g. 0.1% glutaraldehyde).
fixation with a low to moderate concentration of agent to provide moderate stability.
fixation with a moderate to high concentration of agent to provide long term stability.